Project Summary Membrane tension governs endocytosis, exocytosis, cell migration, mitosis, meiosis, membrane transportation, and many other biological phenomena, but there are no tools to map and measure membrane tension in vivo and in real time. Here, we seek funds to demonstrate the feasibility of a novel small molecule imaging probe/contrast agent that produces changes in both fluorescence intensity and photoacoustic intensity as a function of membrane tension. We will compare our probe and methods to the current state-of- the-art: fluorescence lifetime and micropipette/tethering analysis. The technical advance provided by photoacoustic imaging is in vivo imaging of membrane tension: Ultrasound waves are not absorbed/scattered as much as photons and thus imaging through 3-5 cm of tissue is routine. The technical advance of fluorescence intensity is ease of use: Unlike fluorescence lifetime, fluorescence intensity can be done with most common microscopes. Our innovation is grounded in the use of novel conjugation switching chemistry and novel photoacoustic imaging for in vivo imaging. Indeed, a probe that offers in vivo imaging of membrane tension could facilitate fascinating new questions about disease and therapy to be addressed in subsequent proposals using this probe: “How is membrane tension distributed across a 3D organ?; How does an organ’s membrane tension change when it encounters a therapeutic?; and How does an organ’s membrane tension change across the lifespan or with stressors?” This exploratory work will use the following aims to test the feasibility of the contrast agent and gain new technical knowledge about its quantitative advances over the state-of-the-art lifetime- and micropipette-based approaches. Aim 1 will synthesize and characterize the probe using a logical yet innovative organic chemistry workflow. Aim 2 will use giant unilamellar vesicles with tunable membrane tension to test the probe versus a commercially available fluorescence lifetime probe. We will use a micropipette to establish the ground truth tension values and then compare the sensitivity of fluorescence lifetime (gold standard) versus fluorescence intensity and photoacoustic intensity via five different vesicle populations with unique membrane tension values. The imaging method with the steepest slope will be the most sensitive—we expect that our probe will be more sensitive than lifetime because of its activatable nature. Aim 3 will test the novel probe with cultured cells. We will control the membrane tension via osmotic pressure and compare the imaging data of cells at hypo, hyper, and isotonic conditions. To validate the in vivo utility of photoacoustic imaging, we will image cells locked into high or low membrane tension states beneath increasingly thick pieces of tissue-mimicking materials—this experiment will be a proof of concept of in vivo imaging of tension differences. This work is feasible because of Jokerst’s expertise in contrast agent development and in vivo imaging. All needed tools and personnel are in place, and the work is ideally responsive to RFA-22-126’s call for conceptual studies in technology development.

Project Narrative: The cell membrane tension dictates the shape and many downstream properties of cells and the organs made of cells; thus, membrane tension is important in disease but difficult to measure. Here, we propose to build a contrast agent that can map and measure the cell membrane tension using an optical or ultrasound signal. This contrast agent will help guide the detection and treatment of important diseases and be a tool for in vivo research.